Principal Investigator/Program Director (Last, first, middle): HAAS, ARTHUR, L RESEARCH &RELATED Other Project Information 1. * Are Human Subjects Involved? m Yes l No 1.a. If YES to Human Subjects Is the IRB review Pending? m Yes m No IRB Approval Date: Exemption Number: 1 2 3 4 5 6 Human Subject Assurance Number 2. * Are Vertebrate Animals Used? m Yes l No 2.a. If YES to Vertebrate Animals Is the IACUC review Pending? m Yes m No IACUC Approval Date: Animal Welfare Assurance Number 3. * Is proprietary/privileged information m Yes l No included in the application? 4.a.* Does this project have an actual or potential impact on m Yes l No the environment? 4.b. If yes, please explain: 4.c. If this project has an actual or potential impact on the environment, has an exemption been authorized or an environmental assessment (EA) or environmental impact statement (EIS) been performed? m Yes m No 4.d. If yes, please explain: 5.a.* Does this project involve activities outside the U.S. or m Yes l No partnership with International Collaborators? 5.b. If yes, identify countries: 5.c. Optional Explanation: 6. * Project Summary/Abstract 7249-Abstract.pdf Mime Type: application/pdf 7. * Project Narrative 8473-Gm34009-22_relevance.pdf Mime Type: application/pdf 8. Bibliography &References Cited 5469-Literature_Cited.pdf Mime Type: application/pdf 9. Facilities &Other Resources 9258-Resources-Facilities.pdf Mime Type: application/pdf 10. Equipment 1452-Resources-Major_Equipment.pdf Mime Type: application/pdf Tracking Number: Other Information Page 5 OMB Number: 4040-0001 Expiration Date: 04/30/2008 Principal Investigator/Program Director (Last, first, middle): HAAS, ARTHUR, L The ubiquitin/26S proteasome pathway is a fundamental strategy for eukaryotic cell regulation in which proteins are targeted for destruction by assembly on them of specific polyubiquitin degradation signals that are recognized by the 19S regulatory complex of the 26S proteasome. A super family of ubiquitin- like proteins that undergo parallel but distinct conjugation reactions have co-evolved to serve regulatory functions distinct from targeted degradation. The functional specificities of these post-translational modificationsrequirepreciselydirectedproteininteractionsamongcomponentsoftheligationmachinery. Duringthecurrentfundingperiodconventionalbiochemicalandgeneticapproacheshavebeenexploited inparttodefinetheseproteininteractionsathighresolution. Studiesproposedforthenextfundingperiod comprise three Specific Aims designed to build upon these advances. Specific Aim 1 will use biochemical and genetic approaches to study E1-catalyzed activation of Class 1 ubiquitin-like proteins andwillexaminetheroleofthecarboxylterminal [unreadable]-graspdomainasaspecificityfilterforcognateE2/Ubc recognition, the role of [unreadable]-grasp domain versus Ubc12 amino-terminal peptide binding in transition state stabilization during Nedd8 activation, and characterize selected E1 residues in binding versus catalytic roles. Specific Aim 2 will examine the mechanism of a novel subset of Bi-functional Ligases capable of modifying their cognate protein substrates with either ubiquitin to target degradation or the interferon- inducedISG15ubiquitin-likeproteintoblockdegradation. KineticstudieswillquantifytheabilityofUbcH7 (ubiquitin specific) and UbcH8 (ISG15 specific) to support Epf/Trim25 ligase in regulating levels of the anti-mitotic 14-3-3[unreadable] protein implicated in breast and prostate cancers. Other studies will examine the consequences of ubiquitin versus ISG15 modification on the half life of 14-3-3[unreadable]. Specific Aim 3 will continue studies on the enzymology of Mdm2 and MdmX homo- and heterodimers as ubiquitin ligases. Kinetic studies will examine the mechanism of MdmX-stimulated Mdm2-dependent ubiquitination, test a two-